Spiders pertaining to the Loxosceles genus are commonly known as violin spiders because they have a violin-shaped mark with the neck pointing backwards in the anterosuperior part of the cephalothorax (Platnick, 2000).
Spiders of this genus are found all over the world, generally in tropical and temperate climate regions (Ramos, 2000). In Mexico, there are about 39 species of this genus (Hoffman, 1976; Gerstch, 1983). In their natural habitat, they can be found under the bark of trees, under rocks and in caves. They can be found in coexistence with human beings: under furniture, in the corners of rooms, in cracks, grooves of livestock facilities, wood, bricks, and abandoned waste. One of the main causes for Loxosceles bite accidents is, precisely, the constant coexistence with man.
Poisoning caused by the bite of spiders of the Loxosceles genus is called LOXOSCELISM. The violin spider bite commonly produces local necrotic lesions or dermonecrosis (necrotic Loxoscelism), while in some cases it can cause non-necrotizing systemic effects (systemic Loxoscelism).
The extent of local necrosis is related to the spider's stage in of development, the dose of venom injected in the bite and the immune state of the patient (Moye de Alba, 1997; Maguire, 1998).
Dermonecrosis is preceded by edema, the accumulation of inflammatory cells and vasodilatation, all of which culminates in a black vesicle commonly called “bull's eye” lesion. Sometimes the Loxosceles genus may also produce intravascular hemolysis associated with spherocytosis, a condition that persists for several days (Maguire, 1988; Rosse, 1998).
In Mexico, 15 cases of violin spider bite poisoning were treated in the Social Security institute (personal information, Dr. Maria del Carmen Sánchez. “La Raza” Hospital, Mexico City) during the past 5 years, 11 in adults and 4 in children. In 53.3% of the cases, in addition to the necrotic loxoscelism, there was systemic loxoscelism, and 62% of the cases where both occurred, the patients died.
Biochemistry of the Venom
Until now, few spider venoms have been studied in detail. The Loxosceles venom is composed of at least ten to twelve components (Russell, 1987), among them: sterases, alkaline phosphatase, hyaluronidase, phosphohydrolases, lypases and proteases, among others. It has so far been proven that the main component, and the one that causes dermonecrosis, is sphingomyelinase D (SMD). This enzyme binds to the cell membranes (epithelial, endothelial of the vascular tissue and red blood cells) hydrolyzing sphingolipids to subsequently release phosphoceramide and choline (Gatt, 1978). The hydrolysis induces the chemotaxis of neutrophils causing vascular thrombosis and an Arthus-Type reaction (Moye de Alba, 1997; Maguire, 1998; Sánchez, 1993).
Hyaluronidase, other enzymes involved in poisoning, are common in the poisons of almost all spiders (Tan and Ponnundarai, 1992). They have been detected in a considerable number of species (Geren, 1984) including Loxosceles sp., although in these last species, very low enzymatic activity has been reported (Wright, 1973). Hyaluronidases are considered venom-dispersion factors, because the hydrolysis of the hyaluronic acid facilitates the diffusion of the other toxic components within the victim's tissues (Cevallos et al., 1992). Hyaluronidases act as a dispersing agent and it is thought that proteases might be directly involved in dermonecrosis through the digestion of the proteins that form the extracellular matrix (Young, 2001).
Recent studies have identified two proteases, Loxolysine A and Loxolysine B, in L. intermedia. Loxolysine A is a 20-28 kDa metalloprotease with fibrogenolytic activity (degrading fibrinogen) and fibronectinolytic activity (degrading fibronectin). This protein might be involved in the local hemorrhagic effects observed at the site of the bite and in some cases in the systemic hemorrhages, while Loxolysine B is a 32-35 kDa protease with gelatinolytic activity, and although its function is as yet unknown, it may possibly participate in the degradation of collagen within the extracellular matrix (Feitosa et al., 1998).
Three isoforms (P1, P2, and P3) of the necrotoxic fraction have been found in L. intermedia; they were extremely similar to each other at the biochemical and immunological level. The first two are necrotoxic, P2 with a greater effect, while P3 was completely inactive. The analysis of the amino acidic sequences of the first 35 amino acids of the extreme amino terminus of the isoforms revealed that they were identical in many ways. They were also compared with the partial sequences of the toxins of other previously reported Loxosceles species, obtaining a high degree of similarity (Tambourgi, 1998).
In 1968 Smith and Micks demonstrated that the injection of L. reclusa, L. laeta or L. rufuscens venom into rabbits produced similar necrotic reactions. Recent studies compared the amino terminus sequences of sphingomyelinase of L. reclusa, L, deserta, L. gaucho, L. intermedia and L. laeta venom determining that they were homologous among them (Barbara et al., 1996B).
To date, the complete sequences of the sphingomyelinase D of only 2 of the Loxosceles species have been reported—L. Laeta (Fernandes Pedrosa et al., 2002) and L. intermedia (Kalapothakis et al., 2002); they show an identity of barely 59% between them. Only the first 34 AAs of the sphingomyelinase of L. reclusa are known and they happen to have an 85.7% similarity with the equivalent sequence of L. intermedia and 60% with that of L. laeta; for this reason it is impossible to establish a probable sequence for the almost 244 AAs of the enzyme that are still unknown. Similarly, only 35 AAs of the amino terminus region of the sphingomyelinase D of L. deserta and 39 of that of L. gaucho are known (for L. deserta only the sequence derived from the gene is known).
Work on the generation of antibodies against particular species of Loxosceles and cross tests of the same with venoms of spiders of other species of this genus have been reported. For example, a set of monoclonal antibodies against the dermonecrotic component (of 35 K da) of the venom of L. gaucho was developed, which, while being effective in recognizing and neutralizing the homologous venom, was far less able to recognize the venoms of L. laeta and L. intermedia. Its neutralizing capacity was almost nil, compared with the polyclonal antibodies generated against the same component of L. gaucho which adequately recognized and neutralized the venom of L. intermedia, and partially (60%) recognized that of L. laeta, suggesting the presence of different epitopes in the dermonecrotic components of these species, as well as differences in the composition and toxicity of these venoms (Guilherme et al., 2001). Moreover there is evidence of a marked cross-reactivity between the venoms of L. reclusa and L. deserta when the venom of either of the two species is used to generate antibodies (Goomez et al., 2001).
In general terms, there are two approaches for the treatment/prevention of poisoning by poisonous animals such as the violin spider: passive immunization (by means of sero-therapic and fab-therapic agents) and active immunization (through vaccines); the former is a therapeutic measure, while the latter is rather a preventative measure.
Both venoms and isolated toxins have been used to generate vaccines. However, exposure to most venoms does not result in protective immunity. Furthermore, all attempts to create protective immunity against venoms, such as vaccines, have failed (Russell, 1971). In contrast, success has been obtained creating this type of immunity against individual toxins, including vaccines against diphtheria (Audibert et al., 1982), tetanus (Alouf, 1985), the toxoid of α-Latrotoxin (Alagón et al., 1998) and sphingomyelinase D of L. laeta (Araujo et al., 2003).
Passive Immunization
Aside from the palliative of some the palliative treatment of some of the specific symptom, the only treatment available for poisoning is passive immunization.
In the case of passive immunization, the antibodies or their fragments that will bind to the venom (antigen) are exogenous; i.e., they are produced in a first animal. The serum or antivenom from the first animal is then administered to the individual already affected by poisoning (host) to provide him with an immediate and active source of specific and reactive antibodies. The administered antibodies or their fragments will work then, in some sense, as if they were endogenous antibodies, binding the toxins of the venom and neutralizing their toxicity.
Depending on their final use, commercial generation of antivenoms can be undertaken, in various mammals such as mice, rabbits, goats, cows and horses, the horse being the animal of choice of moss laboratories since it is sturdy and tolerates the immunization process and especially because it produces high outputs (up to 16 L per bleeding).
However, there are some technical disadvantages to using horses for the production of antivenoms, among them, the need for large amounts of venom (immunogen or antigen) for performing immunization, forcing the laboratories to have large arachnariums or to contract out the work of gathering large collections of specimens in order to have sufficient quantities of venom available. For example, it is estimated that the production, evaluation and quality control of a lot of antivenom in horses requires highly standardized venom from five thousand spiders, which limits its commercial feasibility. Therefore, having recombinant immunogens capable of triggering an immune response comparable to that triggered by the administered venoms may be a significant alternative for the production of antivenoms, since stable and consistent immunogens would be produced in sufficient quantities at significantly lower costs and with fewer risks than those incurred by keeping arachanariums or the impact of massive collections on ecosystems.
In particular, there are two reports about the use of recombinant proteins as immunogens for the generation of antibodies against spider venoms in mammals, namely, the toxoid of α-Latrotoxin (Alagón et al., 1998) and a fusion protein that comprises the sequence of sphingomyelinase D of Loxosceles intermedia (Araujo et al., 2003).
In Mexico and Latin America, one of the main producers of antivenoms against the venoms of snakes and spiders (scorpions and black widow spiders) is the Instituto Bioclón, S. A. de C. V., which produces antibodies in horses and later purifies and hydrolyzes them in such a way that their antivenoms are in fact F(ab′)2 fragments of the antibodies, i.e., they are fabotherapics. Specifically, they produce antivenom against, the venom of the black widow spider, Aracmyn®.
Due to the variety of common and serious side effects of non-purified antivenoms, the physician must be extremely careful to avoid giving excessive amounts of equine products. A generally accepted theory is that the high incidence of side effects from the current horse antivenoms is caused by the excess of irrelevant protein they contain (irrelevant in the sense of not having a specific activity against the venom). According to this theory, the removal of that irrelevant protein could reduce the exogenous protein charge applied to the body and as a consequence, reduce the incidence of adverse immune responses.
Some researchers in the state-of-the-technique have considered the possibility of purification by immunoaffinity. Most of those studies have only tested antibodies against a single toxin; for example, Yang (1977) proved the purification by immunoaffinity of antibodies against a snake venom toxin. This researcher used cobratoxin, a neurotoxic protein isolated from the venom of the Taiwan cobra (Naja naja atra), bound to Sepharose, as an antigenic matrix and used formic acid to elute the toxin-specific antibodies. The antibodies thus purified were reported to have greater ability to neutralize the toxin than the non-purified serum.
Other researchers have been following similar purification schemes, such as Kukongviriyapan et al. (1982), who used the toxin 3 of Naja naja siamensis bound to several materials to form antigenic matrixes, obtaining a separation of horse-specific antibodies; Ayeb and Delori (1984), who also followed Yang's scheme to purify antibodies against scorpion-specific toxins; and Lomonte et al. (1985), who purified antibodies against the myotoxin of B. asper coupled to Sepharose.
Thus again, having recombinant sphingomyelinases that can specifically bind, preferably bound to an inert matrix, only those antibodies or their fragments that have a high specificity towards the necrotoxic component of the violin spider venom, may be of great help for removing the irrelevant protein for the treatment of poisoning, significantly reducing the risk of adverse immune reactions.
After an incident with Loxosceles, it is not uncommon for the treating physician, or even the affected patients or his/her parents in the case of children to, wrongly assume that it is the bite of an insect or of another type of spider, therefore applying insufficient or wrong treatment to the patient. By the time the unequivocal symptoms of loxoscelism start to appear, the necrosis of the tissue surrounding the wound may be quite advanced and can only be corrected using a skin graft. In this sense, it would be convenient to have an easily readable diagnostic system that allows the treating physician to determine during the first hours after the incident whether it is indeed a violin spider bite and to immediately start an effective treatment that prevents the development of necrosis.
Access number in the Gene Bank:species*Lb1.AY559844L. boneti*Lr1.AY559846L. reclusa*Lr2.AY559847L. reclusa*Ll1.—L. laeta*Ll2.—L. laetaLa.AAP44735L. arizonicaLi.AAQ16123L. laetaLl.H17.AAM21154L. laetaLlH13.AAN121155L. laeta*This patent